Nerve transection is the most severe neural injury (Schmidt 2003). Following transection, the proximal segments of nerves in the peripheral nervous system are capable of regenerating to restore nerve function (Rutkowski 2004). Myelin debris is removed and neurotrophic factors are released by Schwann cells and macrophages that guide the regenerating axons and allow for restored function (Huang 2006). The current “gold standard” for repairing transected nerves in the peripheral nervous system is an autologous nerve graft (Lundborg 1988, Meek 2008, Schmidt 2003). However, there are several concerns that arise with nerve grafting. Harvesting the donor nerve graft can lead to donor site neuroma, loss of function, and scarring (Taras 2005). Concerns also arise over the limited supply of nerve graft donors (Millesi 1991). The functional recovery from this type of procedure is also has variable, only approaching 80% on average (Hudson 2000, Chiu 1995).
The drawbacks of autologous nerve grafting have led to an increased focus in neural tissue engineering research. Current research focuses on finding an ideal nerve guidance channel for peripheral nerve repair. Nerve guidance channels help direct regenerating axons with nerve-compatible biomaterials and neurotrophic factors (Hudson 2000). They avoid the need for a second surgery as well as the potential risks involved with nerve grafting, including donor site morbidity (Taras 2005). They also provide additional benefits in that they prevent axon escape and can be used in hard to reach locations (Meek 2008). Several requirements must be considered when manufacturing a nerve guidance channel including shape, biocompatibility, wall porosity, degradation rate, mechanical strength, and material electrical conductivity (Huang 2006). The material must be appropriate to allow for the penetration of regenerating axons while not damaging the axons (Balgude 2001). The need for a longitudinal distribution of axons also makes it difficult to design scaffolds for nerve repair (Wang 2009). These requirements place limits on the types of materials that may be used for nerve repair.
Nerve guidance channels are fabricated using synthetic or natural materials, both of which have specific benefits and drawbacks. Synthetic materials are beneficial because they allow for alteration of various properties, including porosity, mechanical strength, and degradation rate (Schmidt 2003, Hudson 2000). Drawbacks to synthetic materials include biocompatibility, immune rejection, poor cell adhesion, and mediocre tissue repair (Schmidt 2003). Natural materials are beneficial because they are more biocompatible and less toxic (Schmidt 2003, Hudson 2000, Taras 2005). Drawbacks, however, include isolation issues (Schmidt 2003). Unfortunately, there are still several complications that may arise with nerve guidance channels regardless of the material used. Most importantly, guidance channels can only currently be used for distances less than 3 cm and nerve grafts must still be used for large gaps (Kemp 2008, Meek 2008).
Current research in tissue engineering has focused on improving nerve guidance channels to enhance nerve regeneration (Hudson 2000, Wang 2009). Current projects have focused on 6 main conduit adaptations: 1) porous channel walls (Huang 2006), 2) neurotrophic factor release (Piotrowicz 2006), 3) incorporation of Schwann cells (Galla 2004), 4) aligned intraluminal matrix (Lu 2005) and 6) electrical properties (Bryan 2004). All of these methods are still faced with the drawbacks of using scaffolds for tissue engineering as mentioned above.
Current experiments aimed at creating three dimensional (3-D) nerve constructs mainly focus on the use of agarose gels and other hydrogel scaffolds (Bellamkonda 1995). Hydrogels are attractive for scaffolds due to their biocompatibility (Luo 2004). The addition of extracellular matrix and neurotrophic factors such as laminin and nerve growth factor to hydrogel scaffolds have led to enhanced neurite extension (Yu 1999). The factor infused hydrogel can be used to fill nerve guidance channels to enhance neurite growth and allow for the development of 3-D nerve repair (Yu 1999). Matrigel™ has also been shown to promote neural growth but is unappealing due to its tumorigenic origins (Bellamkonda 1995). Collagen and collagen-gycosaminoglycan matrices have also proven successful peripheral nerve repair (Spilker 2001). All of these experiments still involve scaffolds and all of the problems that are associated with the use of scaffolds.
Three dimensional fibroblast constructs and muscle constructs from a tissue monolayer can be fabricated (Calve 2004). Unlike fibroblast and muscle cell monolayers, a nerve monolayer does not roll up into a 3-D construct. Muscle monolayers will roll up due to contracting muscle cells and fibroblasts roll up due to tension developed between the cells in the monolayer. A nerve monolayer, however, has no source of strain between cells and will merely continue to branch out in one plane.
Current technologies related to neural tissue engineering focus on using nerve guidance channels for the repair of peripheral nerve injury (Ray 2009, Deumens 2010). These scaffolded guidance channels are often incorporated with combinations of Schwann cells, stem cells, and neurotrophic factors which direct and enhance growth of regenerating nerve (Hudson 2000. While these engineered conduits have found success with small peripheral nerve defects, they are still faced with several drawbacks associated with the use of scaffolds, including biocompatibility, immune rejection, poor cell adhesion, and mediocre tissue repair. The need for a more biocompatible, readily available engineered conduit for repair of larger defects persists. The experiments conducted here present the technology for the development of a scaffoldless three-dimensional engineered neural conduit from a readily available, biocompatible source of cells, adipose-derived stem cells (ASC), which overcomes the drawbacks associated with current autograft and scaffold-based technologies.
Previous work indicated that neural cells would proliferate and form a network of cells across an established monolayer of muscle (Larkin 2006). The objective of the experiments described herein was to use similar technologies to grow a nerve monolayer on an existing fibroblast monolayer so that when the fibroblast monolayer rolls up due to tension between the fibroblast cells, a 3-D construct with an external fibroblast sheath and an internal core of interconnected nerve cells would form. To accomplish this, nerve cells suspended in neural basal medium (NBM) were seeded on a confluent monolayer of fibroblasts. Once the neural cells had proliferated and migrated across the fibroblast monolayer, the media is changed to stimulate the fibroblast monolayer to roll up into a 3-D construct effectively forming a 3-D fibroblast-nerve construct as a result. These 3-D fibroblast nerve constructs may be used for surgical repair of nerve transection.